CN117384927A - 番茄LecRK26基因在调控植物灰霉病抗性中的应用 - Google Patents
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Abstract
本发明公开了番茄LecRK26基因在调控植物灰霉病抗性中的应用,涉及基因工程技术领域,具体体现为在提高番茄果实对灰霉病抗性、降低番茄果实灰霉病发病率以及病斑大小中的应用。在番茄中过表达凝集素类受体蛋白激酶基因LecRLK26的步骤如下:首先通过构建含LecRLK26基因的植物重组过表达载体,然后将该载体转入番茄后得到较非转基因植株有显著表达的LecRLK26转基因阳性番茄植株。转基因番茄的果实对灰霉病抗性显著强于野生型植株,发病率和病斑大小显著低于野生型。与传统育种方法相比,本发明采用基因工程技术,高效并可稳定遗传地提高了番茄果实对灰霉病的抗性,有效缓解了灰霉病对植物的危害问题。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及到番茄LecRK26基因在调控植物灰霉病抗性中的应用。
背景技术
灰霉病是由灰葡萄孢(Botrytis cinerea)侵染引起的真菌病害之一,侵染范围十分广泛,包括茄科、葫芦科、蔷薇科等植物的根、茎、叶和果实均可发病,危害作物产量和品质。目前,灰霉病的防治主要以化学方法为主,如使用多菌灵等物质。但长期化学药物的使用易使病菌产生抗药性,同时也会对植物生长造成一定影响,影响防治效果。同时,大量化学药物的使用还会加大对环境和人体的危害。近年来,随着基因工程的迅速发展和技术的日臻完善,转基因技术在培育抗性植物品种方面展示了巨大的潜力,采用转基因的方法提高植物对灰霉病的抗性是有效控制灰霉病危害的措施。凝集素类受体样蛋白激酶在植物病害防御中的报道较多,但在番茄中的报道很少。本研究通过基因工程的方法,从番茄中克隆了一个灰霉病抗性基因LecRLK26,在番茄中过表达LecRLK26基因增强了番茄果实对灰霉病的抗性。表明番茄LecRLK26基因有提高植物对灰霉病抗性的能力,具有较大的应用价值。
发明内容
针对现有技术中的上述不足,本发明提供了番茄LecRK26基因在调控植物灰霉病抗性中的应用,高效并可稳定遗传地提高了番茄果实对灰霉病的抗性,番茄凝集素受体样蛋白激酶LecRLK26基因过表达番茄果实对灰霉病抗性更高,发病率和病斑大小显著降低,有效缓解了灰霉病对植物的危害问题。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:采用基因工程的方法,在番茄中过表达LecRLK26基因。
番茄LecRLK26基因作为正调控基因在如下(1)-(4)至少一项中的应用:
(1)提高番茄果实对灰霉病的抗性;
(2)降低番茄果实灰霉病的发病率;
(3)降低番茄果实灰霉病的病斑大小;
(4)培育高抗灰霉病番茄品种。
上述番茄LecRLK26基因的核苷酸序列包括如SEQ ID NO.1所示,以及除SEQ IDNO.1以外的编码如SEQ ID NO.2所示氨基酸序列的核酸分子。
上述番茄LecRLK26基因所调控的番茄凝集素受体样蛋白激酶LecRLK26作为正调控因子在如下(1)-(4)至少一项中的应用:
(1)提高番茄果实对灰霉病的抗性;
(2)降低番茄果实灰霉病的发病率;
(3)降低番茄果实灰霉病的病斑大小;
(4)培育高抗灰霉病番茄品种。
上述番茄凝集素受体样蛋白激酶LecRLK26的氨基酸序列如SEQ ID NO.2所示,以及列经取代、缺失和/或添加一个或多个氨基酸,且表达相同功能蛋白质的氨基酸序列。
含有番茄LecRLK26基因的重组表达载体、转基因细胞系或者工程菌在如下(1)-(4)项中的应用:
(1)提高番茄果实对灰霉病的抗性;
(2)降低番茄果实灰霉病的发病率;
(3)降低番茄果实灰霉病的病斑大小;
(4)培育高抗灰霉病番茄品种。
上述含有番茄LecRLK26基因的重组表达载体的核苷酸序列如SEQ ID NO.3所示。
一种利用番茄LecRLK26基因提高番茄果实对灰霉病抗性的方法,使番茄LecRLK26基因过量表达。
进一步,使番茄LecRLK26基因过表达的具体步骤包括:外源转入番茄LecRLK26基因;或者上调番茄基因组中原有的LecRLK26基因的表达。
一种利用番茄LecRLK26基因培育高灰霉病抗性的番茄品种的方法,包括以下步骤:将番茄LecRLK26基因转入番茄野生型植株中,获得LecRLK26基因过量表达且稳定遗传的番茄,即番茄转基因植株。
上述番茄转基因植株与番茄野生型植株相比具有如下(1)-(3)至少一项特点:
(1)转基因番茄对灰霉病的抗性显著高于野生型番茄;
(2)转基因番茄灰霉病的发病率显著低于野生型番茄;
(3)转基因番茄灰霉病的病斑大小显著小于野生型番茄。
进一步,上述将番茄LecRLK26基因转入野生型番茄中的方法为聚乙二醇法、农杆菌浸染法或基因枪法。
综上所述,本发明具有以下有益效果:
1、本发明通过在番茄中过表达凝集素类受体蛋白激酶LecRLK26基因,并构建含LecRLK26基因的植物重组表达载体,将该载体转入番茄后所得转基因番茄阳性植株中LecRLK26基因较非转基因植株有显著更高的表达。转基因番茄的果实对灰霉病抗性显著强于野生型植株,发病率和病斑大小显著低于野生型。
2、与传统育种方法相比,本发明采用基因工程技术,高效并可稳定遗传地提高了番茄果实对灰霉病的抗性,为番茄抗性育种做出了积极的探索,具有较好的市场前景和经济价值。
附图说明
图1为LecRLK26过表达转基因株系的基因相对表达量;
图2为LecRLK26过表达转基因株系接种灰霉病菌7d和14d后的表型;
图3为LecRLK26过表达转基因株系接种灰霉病菌7d后的发病率;
图4为LecRLK26过表达转基因株系接种灰霉病菌7d后的病斑直径。
具体实施方式
下面结合实施例对本发明进行进一步的说明,但并不构成对本发明的限定。
本发明实施例中使用的番茄品种为“Solanum lycopersicum L.cv.Micro Tom”,为实验室留种。8GWN中间载体、K303载体为实验室改造,大肠杆菌DH5α和根癌农杆菌GV3101由本实验室保存;RNA提取试剂盒(Reagent)由索莱宝公司提供;Taq DNA聚合酶由重庆葆光生物科技有限公司提供;cDNA合成试剂盒(PrimeScriptTM RT reagent Kitwith gDNA Eraser)、限制性内切酶、PrimeSTAR高保真DNA聚合酶均购自TaKaRa公司;同源重组酶(/>II One Step Cloning Kit-C112)由南京诺唯赞生物科技股份有限公司提供;Gateway LR Clanase酶购买自赛默飞世尔科技公司;DNA提取和纯化试剂盒(离心柱型)为OMEGA公司产品;其余试剂均为分析纯试剂。
实施例1LecRLK26基因的全长序列克隆
(1)设计引物:以番茄(Solanum lycopersicum L.)凝集素类受体蛋白激酶LecRLK26(Solyc10g006710.3.1)的cDNA序列为模板,设计引物克隆番茄LecRLK26基因的全长CDS序列,同时根据中间载体8GWN在引物的两端引入同源重组片段,引物分别为RK15-F和RK15-R,序列如下:
RK15-F:5'-atacttccaactagtgcggccgc tattaccacgacaaacacc-3'(SEQ IDNo.4);
RK15-R:5'-atggtcatcccgggacctgcagg tcgagcgtccaacaaagtga-3'(SEQ IDNo.5);
(2)PCR反应:使用RNA提取试剂盒(Reagent)提取番茄叶片总RNA,再根据cDNA合成试剂盒(PrimeScriptTM RT reagent Kit with gDNA Eraser)的使用说明合成cDNA,再以cDNA为模板,以SEQ ID No.4和SEQ ID No.5为引物,扩增带有同源片段的LecRLK26基因全长。PCR反应体系为如表1所示。
表1 PCR反应体系
PCR反应程序为:98℃预变性3分钟;然后98℃变性15秒,55℃退火15秒,72℃延伸2分钟,共35个循环;最后72℃后延伸5分钟。
(3)制备重组质粒8GWN::35S-LecRLK26-GFP:将PCR扩增产物经琼脂糖凝胶电泳鉴定,结果显示,扩增获得约2500bp的目的条带,然后将扩增产物采用DNA纯化试剂盒进行回收纯化,将纯化的LecRLK26基因全长片段与8GWN载体通过同源重组酶(IIOne Step Cloning Kit-C112)进行连接,获得重组质粒8GWN::35S-LecRLK26-GFP。再将上述质粒转化DH5α大肠杆菌后,鉴定阳性克隆,并委托生工生物工程股份有限公司进行测序。结果显示,LecRLK26基因全长为2554bp,核苷酸序列如SEQ ID No.1所示。
实施例2构建LecRLK26基因的过表达载体
使用Gateway重组克隆技术将8GWN::35S-LecRLK26-GFP质粒与K303空质粒进行LR克隆,反应体系如下:
20μL的反应体系中,加入8GWN::35S-LecRLK26-GFP质粒150ng、K303空质粒150ng、5×LR Clonase Reaction Buffer 4μL、LR Clonase enzyme mix 4μL、TE buffer加至20μL,混匀后25度60min,完成后加入2μL Proteinase K solution于37度保持10min终止反应。然后取10μL反应产物转化DH5α大肠杆菌后,鉴定阳性克隆。然后挑选单菌落扩大培养后提取质粒,委托生工生物工程股份有限公司进行测序,如果序列正确即证明K303::35S-LecRLK26-GFP载体构建成功。
实施例3将重组质粒K303::35S-LecRLK26-GFP转化农杆菌
(1)构建重组表达菌株:取5μL质粒转入根癌农杆菌GV3101感受态细胞中,在含有质量分数为1.5%的琼脂、50mg/L利福平和50mg/L卡那霉素的YEB固体培养基(pH7.2)中涂板,28±1℃黑暗条件下倒置培养2-3天至长出单菌落,挑取3-5个单菌落做菌落PCR。K303::35S-LecRLK 26-GFP载体用K303F、SC15R引物检测,产物长度为1600bp左右。
K303::35S-LecRLK26-GFP质粒的检测引物序列如下:
K303F:5'-cgtcttgcgcactgatttga-3'(SEQ ID No.6)
SC15R:5'-gctccactccatctaagaccattcc-3'(SEQ ID No.7)
反应体系如表2所示。
表2反应体系
反应条件为95℃预变性3min,95℃变性15s,55℃退火30s,72℃延伸30s,循环数为25个,72℃终延伸5min。
选择阳性菌落用含有50mg/L利福平和50mg/L卡那霉素的YEB液体培养基(pH7.2),在28±1℃、黑暗、200rpm条件下摇床培养1.5天至菌液均匀且OD600为0.8-1.0,然后28±1℃离心弃上清,菌体用新鲜的YEB液体培养基洗涤,再用含有质量分数为3%的蔗糖、pH为5.8的MS培养基100mL重悬,制得农杆菌工程菌液。
(2)转化番茄子叶:用体积分数为75%的酒精浸泡番茄种子1min,无菌水冲洗6次;再用1%(有效氯浓度)的NaClO水溶液浸泡种子10min,无菌水冲洗7次;无菌水浸泡种子4h,播种于MS固体培养基上,光照培养箱26℃(16h光照)/18℃(8h黑暗)培养直至子叶展平,切取已展平的子叶即得番茄外植体。
将番茄外植体的子叶切下,放置在含有3wt%的蔗糖、0.8wt%的琼脂、1mg/L吲哚乙酸、1mg/L玉米素、pH为5.8的MS固体培养基中,25℃(16h光照)/20℃(8h黑暗)预培养1d后置于农杆菌工程菌液中浸染15min后,放回上述含有3wt%的蔗糖、1wt%的琼脂、1mg/L吲哚乙酸、1mg/L玉米素、pH为5.8的MS固体培养基中,在28±1℃黑暗条件下共培养48小时,再转入含有3wt%的蔗糖、1wt%的琼脂、1.0mg/L吲哚乙酸、1mg/L玉米素、200mg/L替卡西林钠克拉维酸钾、120mg/L阿莫西林钠克拉维酸钾和100mg/L卡那霉素、pH为5.8的MS固体培养基中,在25±1℃、光周期16h/d、光照强度3000~5000lx条件下培养至长出愈伤组织及抗性芽;将长3~4cm的抗性芽切下,转入含有3wt%的蔗糖、1wt%的琼脂、200mg/L替卡西林钠克拉维酸钾、120mg/L阿莫西林钠克拉维酸钾和50mg/L卡那霉素、pH为5.8的MS固体培养基中,在25±1℃、光周期16h/d、光照强度3000~5000lx条件下培养至生根,即得转基因番茄植株。
实施例4转基因番茄阳性植株的筛选
1.PCR检测LecRLK26基因是否转入番茄植株中
根据载体K303::35S-LecRLK26-GFP中的基因序列,设计如下特异检测引物:
35SF:5'-atgacgcacaatcccactatccttc-3'(SEQ ID No.8);
SC15R:5'-gctccactccatctaagaccattcc-3'(SEQ ID No.7)。
分别取转基因番茄植株和非转基因番茄植株,提取基因组DNA,再以所得基因组DNA为模板、SEQ ID No.7和SEQ ID No.8所示序列为引物进行PCR扩增,检测LecRLK26基因是否转入番茄植株中。PCR产物经琼脂糖凝胶电泳分析,阳性转基因番茄植株的PCR产物在858bp位置出现目标DNA条带,经测序证明与LecRLK26基因片段序列一致,证实这些植株为转基因番茄阳性植株。再将F1代杂合过表达转基因番茄植株经过2代筛选,即可得纯合过表达转基因番茄。
2.qPCR检测LecRLK26基因在转基因番茄阳性植株中的表达
根据LecRLK26基因序列和番茄内参基因SlActin序列,设计如下引物:
qRKC15-F:5'-gacctcaggcagctatcggaatca-3'(SEQ ID No.9);
qRKC15-R:5'-cg aagaatcaggcagactccaacc-3'(SEQ ID No.10);
qActin-F:5'-tgggtgtgcctttctgaatg-3'(SEQ ID No.11);
qActin-R:5'-gctaagaacgatggacctaatg-3'(SEQ ID No.12)。
分别取转基因番茄阳性植株和非转基因番茄植株的幼叶,提取总RNA,将总RNA反转录为cDNA,再以所得cDNA为模板,分别以qRKC15-F和qRKC15-R,qActin-F和qActin-R为引物进行qPCR,检测LecRLK26基因在转基因番茄阳性植株中的表达量,结果如图1所示。
由图1可知,过表达转基因番茄株系(OE-204、OE-214、OE-219)中LecRLK26基因表达量发生了显著改变。
实施例5过表达转基因番茄阳性植株的表型分析
将实施例4中筛选出的过表达转基因番茄和野生型番茄植株按常规方法培育,并取橙色果实在果实四面接种灰霉病菌(B.cinereal,浓度为2x10-5孢子/mL),每个株系接种5个果实,放置在种子发芽盒中半密闭贮藏,每个株系接种5个生物学重复共接种25个番茄果实。7天和14天后观察和统计,结果如图2-4所示。
由图2-4可知,过表达转基因番茄(OE-204、OE-214、OE-219)灰霉病的发病率和病斑大小显著低于野生型番茄果实。
由此可知,本发明获得的LecRLK26基因过表达番茄的果实对灰霉病的抗性具有显著提升,对番茄抗性育种具有显著的经济价值。
虽然结合附图对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可做出的各种修改和变形仍属本专利的保护范围。
Claims (10)
1.番茄LecRLK26基因作为正调控基因在如下(1)-(4)至少一项中的应用:
(1)提高番茄果实对灰霉病的抗性;
(2)降低番茄果实灰霉病的发病率;
(3)降低番茄果实灰霉病的病斑大小;
(4)培育高抗灰霉病的番茄品种。
2.如权利要求1所述的应用,其特征在于,所述番茄LecRLK26基因的核苷酸序列如SEQID NO.1所示,以及除SEQ ID NO.1以外的编码如SEQ ID NO.2所示氨基酸序列的核酸分子。
3.如权利要求1所述的应用,其特征在于,所述番茄LecRLK26基因所调控的番茄凝集素受体样蛋白激酶LecRLK26作为正调控因子在如下(1)-(4)至少一项中的应用:
(1)提高番茄果实对灰霉病的抗性;
(2)降低番茄果实灰霉病的发病率;
(3)降低番茄果实灰霉病的病斑大小;
(4)培育高抗灰霉病的番茄品种。
4.如权利要求3所述的应用,其特征在于,所述番茄凝集素受体样蛋白激酶LecRLK26的氨基酸序列如SEQ ID NO.2所示,以及列经取代、缺失和/或添加一个或多个氨基酸,且表达相同功能蛋白质的氨基酸序列。
5.含有权利要求1所述的番茄LecRLK26基因的重组表达载体、转基因细胞系或者工程菌在如下(1)-(4)项中的应用:
(1)提高番茄果实对灰霉病的抗性;
(2)降低番茄果实灰霉病的发病率;
(3)降低番茄果实灰霉病的病斑大小;
(4)培育高抗灰霉病的番茄品种。
6.如权利要求5所述的含有番茄LecRLK26基因的重组表达载体,其特征在于,所述重组表达载体的核苷酸序列如SEQ ID NO.3所示。
7.一种利用权利要求1所述的番茄LecRLK26基因提高番茄果实对灰霉病抗性的方法,其特征在于,使番茄中LecRLK26基因过量表达。
8.如权利要求7所述的利用番茄LecRLK26基因提高番茄果实对灰霉病抗性的方法,其特征在于,所述使番茄LecRLK26基因过表达的具体步骤包括:外源转入番茄LecRLK26基因;或者上调番茄基因组中原有的LecRLK26基因的表达。
9.一种利用权利要求1所述的番茄LecRLK26基因培育高灰霉病抗性番茄品种的方法,其特征在于,包括以下步骤:将番茄LecRLK26基因转入番茄野生型植株中,获得LecRLK26基因过量表达且稳定遗传的番茄。
10.如权利要求9所述的利用番茄LecRLK26基因培育高灰霉病抗性的番茄品种的方法,其特征在于,所述将番茄LecRLK26基因转入野生型番茄中的方法为聚乙二醇法、农杆菌浸染法或基因枪法。
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